The invention relates to treating Dutch elm disease by administering to elm trees an elicitor obtained from a Dutch elm disease-causing fungus.
Since its introduction from Europe during the first half of the twentieth century, Dutch elm disease (DED) has decimated North American elm tree populations, the American elm (Ulmus americana L.) being particularly susceptible to DED.
DED is known to be caused by the fungus Ophiostoma ulmi sensu lato (O. ulmi), which is transported between elm trees by the native and European elm bark beetle. The beetle forms tunnels, also known as galleries, in the bark of the elm tree, and leaves spores of O. ulmi in these tunnels. The fungus then spreads through the tree""s water-conducting tubes (vessels). The observable symptoms of DED, namely wilting, yellowing and loss of leaves, and eventually death, are believed to be caused by toxins released by the fungus. One such toxin, which has been associated with DED-like symptoms in American elms, is cerat-oulmin (CU).
Numerous approaches have been tried over the years to eradicate or prevent the spread of DED in elm populations.
One approach has been to control elm bark beetle populations through the use of pesticides or by cutting infected limbs from elm trees. Another approach is to control or inhibit growth of the fungus by treating infected trees with fungicides or less commonly with antagonistic organisms such as bacteria.
However, all of these approaches have disadvantages which limit their effectiveness. In particular, the use of large amounts of chemical pesticides and fungicides is undesirable from an environmental standpoint, particularly in urban areas.
Another approach has been to develop strains of elm trees which are resistant to DED, for example by selective breeding. However, such approaches are typically time consuming and do nothing to prevent the spread of DED in existing elm populations. Furthermore, until recently little was known about the mechanisms of DED resistance in elm trees or the means by which O. ulmi kills its host. Therefore, it was unclear whether or not long-term resistance could be bred into elm trees.
Furthermore, the importance of the American elm lies in its umbrella-shaped crown, which makes it a particularly effective shade tree. No other species of elm can compete with the American elm in this respect. Therefore, developing resistance by cross-breeding the American elm with resistant species of elms is useless if the form of the American elm is not maintained.
None of the above approaches has been completely successful in treating or controlling the spread of DED. Therefore, remaining elm populations remain at risk of being decimated by DED.
Recent research has shown that the American elm, which is particularly susceptible to DED, nevertheless produces a defence reaction when infected by a DED-causing fungus. Specifically, it has been shown that elm trees infected with DED produce several sesquiterpene quinones possessing antifungal properties, these compounds being known collectively as xe2x80x9cmansononesxe2x80x9d, Dumas et al., Experientia 39 (1983), pp. 1089-1090. The mansonones known as mansonones xe2x80x9cAxe2x80x9d, xe2x80x9cCxe2x80x9d, xe2x80x9cDxe2x80x9d, xe2x80x9cExe2x80x9d, xe2x80x9cFxe2x80x9d and xe2x80x9cGxe2x80x9d have all been shown to inhibit the growth of strains of O. ulmi. The structural formulas of these mansonones are shown below. 
Mansonone accumulation in elms is believed to be triggered by specific compounds produced by O. ulmi which are recognized by the elm tree after it is infected by the fungus. These compounds which cause mansonone accumulation are commonly referred to as xe2x80x9celicitorsxe2x80x9d. Mansonone-inducing elicitors are present in the culture filtrate, cytoplasm and cell walls of O. ulmi and have been shown to induce production of mansonones in elm tissue cultures, Yang et al., Eur. J. For. Path. 23 (1993) 257-268, Can. J. Bot. 67 (1989) 3490-3497, and Mycol. Res. 98(3): 295-300 (1994).
Although all strains of O. ulmi produce elicitors, it has been found that the less virulent, xe2x80x9cnon-aggressivexe2x80x9d, strains of O. ulmi cause elm tissue to accumulate mansonones more quickly and in larger amounts than virulent, xe2x80x9caggressivexe2x80x9d, strains of O. ulmi (often referred to as Ophiostoma novo-ulmi). This is consistent with the observation that, although all strains can kill susceptible elm trees, the progress of the disease is slower in trees infected by non-aggressive isolates.
Several mechanisms have been proposed to explain the higher virulence of aggressive strains of O. ulmi. It is believed that differential elicitation and/or suppression of mansonone production in elms is at least partially responsible for the higher level of pathogenicity of aggressive strains of O. ulmi. Therefore, it appears that aggressive strains of the fungus may at least partially suppress the production of mansonones in elm trees.
Attempts have been made to use this difference in virulence to induce resistance to highly virulent strains of O. ulmi in susceptible elm trees. Some early inoculation trials using elm seedlings and elm tissue cultures were encouraging. For example, see, Hubbes and Jeng, Eur. J. For. Path. 11 (1981) 257-264, and Hubbes, Naturaliste can. (Rev. Ecol. Syst.), 115: 157-161 (1988). However, a more recent study conducted with European and hybrid elms concluded that, although there is some benefit to be derived from preventatively inoculating elms with O. ulmi or other fungi, there is little reason to think that the method has immediate promise for the control of DED, Sutherland et al., Eur. J. For. Path. 25 (1995) 307-318.
Therefore, extensive research has been conducted into the defence reactions of elms to DED-causing fungi. However, this research has thus far not resulted in any treatments for DED capable of being successfully used on a widespread basis.
The inventor has overcome the problems in the prior art by inventing a treatment for DED which comprises a method for inducing resistance to DED in DED-susceptible elm trees. The treatment of the invention may be used preventatively to induce DED-resistance in uninfected elm trees, or may be used to treat elm trees which have been infected with a DED-causing fungus.
The method of the present invention comprises administering to a DED-susceptible elm tree an amount of an elicitor obtainable from a DED-causing fungus. Preferably, the method of the present invention utilizes a novel elicitor isolated from the culture filtrate of O. ulmi and having an amino acid sequence identified by the inventor.
The elicitors of the present invention, when administered to a DED-susceptible elm tree, cause a defence reaction in the tree which inhibits the growth of DED-causing fungi. When used as a preventative treatment, the defence reaction allows the tree to resist subsequent infection by a DED-causing fungus such as O. ulmi. One of the most obvious responses making up this defence reaction is the accumulation of mansonones by the tree.
Therefore, the DED treatment according to the invention mobilizes the elm tree""s defence mechanism to protect it from present or future infection by a DED-causing fungus. The elicitors of the invention are non-toxic and stable and may easily be produced in large quantities from cultures of O. ulmi. Furthermore, only a small quantity of elicitor is required to trigger a defence reaction in an elm tree. The small quantities required may be easily administered without damaging the tree.
Therefore, the treatment according to the present invention provides significant advantages over known treatments, which typically require administration to the tree of large quantities of toxic materials either through a large number of holes in the tree or into the tree""s root system.
Therefore, it is one object of the present invention to provide a treatment for DED utilizing elicitors obtainable from DED-causing fungi.
It is another object of the present invention to provide a preventative treatment for DED utilizing elicitors obtainable from DED-causing fungi.
It is yet another object of the present invention to provide a treatment for DED utilizing a novel elicitor isolated from culture filtrates of O. ulmi. 
It is yet another object of the present invention to provide a treatment for DED which utilizes the elm tree""s natural defence reaction to DED-causing fungi.
It is yet another object of the present invention to provide a treatment for DED which causes the accumulation of fungistatic compounds such as mansonones in elm trees.
Accordingly, in one aspect, the present invention provides a method for inducing resistance to Dutch elm disease (DED) in a DED-susceptible elm tree, comprising administering to the tree a glycoprotein elicitor in an amount sufficient to cause a defence reaction in the tree. The defence reaction comprises accumulation of fungal inhibitory compounds in tissue of the tree, and the elicitor is obtainable from a DED-causing fungus.
Preferably, the elicitor is obtainable from the cell interior, cell wall or culture filtrate of a DED-causing fungus, the preferred DED-causing fungus being Ophiostoma ulmi (O. ulmi), most preferably a non-aggressive strain of O. ulmi. 
The elicitor is preferably a glycoprotein obtained from a culture filtrate of O. ulmi strain Q412, and having an amino acid sequence which includes Seq. ID No. 1, described below, which preferably begins at the N-terminal of the amino acid sequence of the elicitor. The molecular weight of the elicitor is preferably at least about 21 kDa.
Preferably, the fungal inhibitory compounds accumulated by the elm tree are mansonones selected from the group comprising mansonones A, C, D, E, F and G. Although the defence reaction comprises accumulation of mansonones, it also preferably comprises lignification and release of hydrogen peroxide.
Preferred DED-susceptible elm trees to be treated according to the present invention are selected from the group comprising Ulmus americana L., Ulmus thomassii Sarg., Ulmus rubra Muhl., Ulmus carpinifolia Gleditsch., Ulmus glabra Huds., Ulmus procera Salisb. and Ulmus laevis Pall., and DED-susceptible cultivars thereof. The most preferred DED-susceptible elm tree is Ulmus americana L.
The amount of elicitor effective to cause the tree to exhibit a defence reaction is preferably from about 5 mg to about 150 mg. In one preferred aspect of the present invention, administering of the elicitor to the elm tree comprises injection of a liquid composition containing the elicitor into the tree, the liquid composition preferably comprising an aqueous solution of the elicitor in a preferred concentration of from about 0.1 mg/mL to about 5 mg/mL. Preferably, the injection delivers the liquid composition inside the vascular system adjacent to the bark of the tree.
In another preferred aspect of the invention, administering of the elicitor to the elm tree comprises insertion of the elicitor in a solid form into the tree, the solid form of the elicitor preferably comprising a solid composition comprising the elicitor, and which is preferably contained in a capsule. The solid composition may preferably additionally comprise acceptable fillers and carriers. Insertion of the elicitor into the tree preferably comprises drilling a hole through the bark of the tree, and inserting the capsule into the hole so that the elicitor is received inside the vascular system adjacent to the bark of the tree.
In another aspect, the present invention provides a program for prevention of Dutch elm disease (DED) in a DED-susceptible elm tree, comprising annual treatment of the tree according to the method of the invention.